Methods of diagnosing renal salt wasting syndrome and alzheimer&#39;s disease and methods of treating the same

ABSTRACT

A method is described to diagnose (1) renal salt wasting syndrome and (2) Alzheimer&#39;s disease among dementia patients by measuring a patient&#39;s level of prostaglandin D 2  synthase. Methods are also described to (1) treat renal salt wasting syndrome, (2) inhibit the rate of apoptosis or (3) prevent the onset of, or slow the rate of, progression of Alzheimer&#39;s disease. These methods involve inhibiting the rate of −Δ 12 prostaglandin J 2  synthesis or by inhibiting the activity of −Δ 12 prostaglandin J 2 .

RELATED APPLICATION

[0001] This application is a divisional of U.S. patent application Ser.No. 09/096,335, filed Jun. 11, 1998, which is hereby incorporated byreference.

[0002] Throughout this application, various references are referred towithin parentheses. Disclosure of the publications in their entirety arehereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains.

FIELD OF THE INVENTION

[0003] The present invention relates to a method of (1) diagnosing orassessing the likelihood that a patient is afflicted with renal saltwasting syndrome and (2) diagnosing or assessing the likelihood that apatient is afflicted with or will develop Alzheimer's disease. Thepresent invention also relates to methods of (1) treating, preventingthe onset or slowing the rate of progression of Alzheimer's disease, (2)treating or preventing onset of renal salt wasting syndrome, and (3)inhibiting apoptosis.

DESCRIPTION OF THE RELATED ART

[0004] A new medical syndrome, the renal salt wasting syndrome has beendescribed in patients suffering from pneumonia, cancers of the lung, andbrain diseases such as primary or secondary tumors, brain hemorrhage,AIDS, and Alzheimer's disease (J. K. Maesaka et al. Life Sci. 52:1875,1993, J. K. Maesaka et al., J. Am. Ger. Soc. 41:501, 1993). Patientssuffering from renal salt wasting syndrome have low serum sodium(hyponatremia) and low serum uric acid levels (hypouricemia). Thesepatients share low serum uric acid concentrations and a renal tubulartransport defect for uric acid which results in an increase in thefractional excretion of uric acid. Renal salt wasting syndrome mimicsthe syndrome of inappropriate secretion of antidiuretic hormone (SIADH)in many clinical parameters except that renal salt wasting syndrome hasdiminished total body water and sodium. Total body fluids are increasedin SIADH and decreased in the renal salt wasting syndrome. Because it isextremely difficult to assess accurately the fluid status of patientsthat do not suffer from edema, renal salt wasting syndrome patients arefrequently misdiagnosed as having SIADH.

[0005] The importance of making a differentiation between renal saltwasting syndrome and SIADH is the difference in treatment modalities.SIADH is usually treated with water restriction whereas the renal saltwasting syndrome patients require variable amounts of fluid and saltsupplementation depending on the extent of their salt and waterdeficits. Moreover, large volumes of salt and fluid, particularly water,actually exacerbate the hyponatremia in patients with SIADH which canlead to coma and convulsions. On the other hand, fluid restrictions, acommon treatment for SIADH, could worsen the clinical condition of thepatient with renal salt wasting syndrome because it exacerbates theirunderlying depletion of body fluids.

[0006] Volume depletion and persistence of the hypouricemia andincreased fractional excretion (FE) of urate by the kidneys aftercorrection of the hyponatremia distinguish renal salt wasting syndromefrom the SIADH. Since assessment of extracellular volume (ECV) which isnecessary to determine volume depletion has been shown to be inaccuratein non-edematous and non-ascitic cases (H. M. Chung et al., Am. J. Med.83:905, 1987), it was postulated that it might be possible todifferentiate renal salt wasting syndrome from inappropriate secretionof antidiuretic hormone by scrutinizing urate metabolism and response ofthe patient to saline infusion. However, the necessary salt balancestudies are believed to be less practical than the simple determinationdescribed herein.

[0007] The plausibility of a salt wasting syndrome in patients withneurosurgical or possibly active brain diseases lies in thedemonstration of natriuretic-apoptotic factor(s) circulating in theplasma of patients with neurosurgical and Alzheimer's diseases byMaesaka et al. (Life Sci. 52:1875, 1993; J. Am. Ger. Soc. 41:501, 1993).There was a fourfold or greater increase in apoptosis in culturedLLC-PK1 cells that have been exposed to Alzheimer plasma as compared tonormal and multi-infarct dementia (MID) plasma (J. K. Maesaka et al., J.Am. Soc. Nephrol. 6:740, 1995 (abst.)). However, the identity of thisfactor is not known and the testing of its presence based on an increasein apoptosis in tissue cultured cells is impractical.

SUMMARY OF THE INVENTION

[0008] It is an object of the present invention to provide effectivemethods and kits for diagnosing Alzheimer's disease and renal saltwasting syndrome.

[0009] It is another object of the invention to provide a method oftreating, reducing the risk of onset of, or slowing the rate ofprogression of, Alzheimer's disease.

[0010] It is yet another object of the invention to provide a method totreat or reduce onset of renal salt wasting syndrome.

[0011] It is yet another object of the invention to provide a method toinhibit the rate of apoptosis.

[0012] It is yet another object of the invention to provide a clinicalkit for the quantification of prostaglandin D₂ synthase levels,preferably for aiding diagnosis of Alzheimer's disease and/or renal saltwasting syndrome.

[0013] In one embodiment, the invention provides a method of diagnosingor assessing the likelihood that a patient is afflicted with renal saltwasting syndrome, said method comprising measuring the level ofprostaglandin D₂ synthase in a sample from said patient.

[0014] In another embodiment, the invention provides a method ofdiagnosing or assessing the likelihood that a patient is afflicted withAlzheimer's disease, said method comprising measuring the level ofprostaglandin D₂ synthase in a sample from said patient.

[0015] In yet another embodiment, the invention provides a method oftreating or reducing the risk of acquiring renal salt wasting syndromein a patient in need of such treatment or reduction, said methodcomprising reducing -Δ¹²prostaglandin J₂ levels or activity thereof insaid patient.

[0016] In yet another embodiment, the invention provides a method ofinhibiting the rate of apoptosis in a patient with elevatedprostaglandin D₂ synthase in the plasma or urine, said method comprisingreducing -Δ¹²prostaglandin J₂ levels or activity in said patient.

[0017] In yet another embodiment, the invention provides the method oftreating or reducing the risk of onset of Alzheimer's disease in apatient in need of such treatment or reduction, said method comprisingreducing -Δ¹²prostaglandin J₂ levels, or activity thereof, in saidpatient, other than by administering a cyclo-oxygenase inhibitor.

[0018] In yet another embodiment, the invention provides a diagnostickit for detecting the presence of prostaglandin D₂ synthase in a sample,said kit comprising antibodies to said prostaglandin D₂ synthase, andmeans for measuring prostaglandin D₂ synthase:anti-prostaglandin D₂synthase immunocomplexes.

[0019] The term “inhibitor” as used herein means any agent which reducesthe normal physiological effect of an already-formed agent, e.g. byaction on the agent itself or by antagonistic effect on a receptor forthat agent. EXCEPTION: As used herein, the term “cyclo-oxygenaseinhibitor” does not include cyclo-oxygenase antibodies.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIG. 1 shows a transmission electron micrograph of LLC-PK1 cellsthat have been exposed to AD plasma for 2 hours prior to fixation. Notecondensed and black nuclei (black arrows). Arrowheads represent smallresidual nuclear bodies engulfed by a neighboring cell. White arrowsshow a section fold. Magnification: A=2200X, B=4500X. This shows thatLLC-PK1 cells undergo apoptosis after exposure to AD plasma.

[0021]FIGS. 2A and 2B illustrate a TUNEL assay with ApoDetek Kit (Enzo).FIG. 2A is a light micrograph of a TUNEL assay performed on LLC-PK1cells exposed to control plasma for 2 hours showing normal pale stainingnuclei. FIG. 2B is a light micrograph of a TUNEL assay performed onLLC-PK1 cells exposed to plasma of patients with Alzheimer's disease for2 hours showing condensed, dark nuclei (large arrowheads) and the normaloval pale nuclei (small arrowheads). Magnification=300X. This shows thatLLC-PK1 cells undergo apoptosis after exposure to AD plasma.

[0022]FIGS. 3A and 3B show a dose response (A) and Time course (B) ofapoptosis in LLC-PK1 cells exposed to plasma of patients withAlzheimer's disease as measured by TUNEL assay. This shows that ADplasma contains a component that causes LLC-PK1 cells to undergoapoptosis.

[0023]FIG. 4 shows internucleosomal DNA cleavage in LLC-PK1 cells thathad been exposed to plasma of patients with Alzheimer's disease (AD) forintervals 2, 3, 4, and 5 days. Maximum DNA fragmentation was 4 daysafter exposure to AD plasma and showed the characteristic 180 bpspacing. Control (NC) plasma did not exhibit a ladder even afterincubation for 5 days. The DNA laddering indicates that apoptosis occursin LLC-PK1 cells exposed to AD plasma.

[0024]FIG. 5 is an elution profile of pooled plasma from patients withAlzheimer's disease (4.5 mL) chromatography on (1×8 cm) Affi-Gel BlueGel Agarose column (20 mM phosphate Buffer, pH 7.1) (Flow Rate=1mL/min.) (15 mL Load and Wash, 25 mL 0.5 M NaCl fraction, 10 mL 2M NaClfraction). Line represents active fraction.

[0025]FIGS. 6A and 6B show the results of iso-electric focusing. FIG. 6Adepicts fractionation of 0.5 M NaCl eluate from Affi-Blue run on Rotofor(BioRad) using pH 3-10 gradient. FIG. 6B depicts fractionation of theactive pool from FIG. 6A (pI 4.4-5.7) on Rotofor, using pH 4-6 gradient.Arrow points to fraction 2, line represents the active fraction.

DETAILED DESCRIPTION OF THE INVENTION

[0026] In accordance with the invention, prostaglandin D₂ synthase is amarker for Alzheimer's disease and is also a marker for renal saltwasting syndrome. Levels of Prostaglandin D₂ synthase are elevated inthe blood and urine of patients suffering from Alzheimer's disease ascompared to normal, non-demented age and gender-matched controls andcomparably demented patients with multi-infarct dementia. The levels ofprostaglandin D₂ synthase are also elevated in the blood and urine ofpatients suffering from renal salt wasting syndrome and not in patientswith the syndrome of inappropriate secretion of antidiuretic hormone(SIADH), a common cause of hyponatremia.

[0027] The bioassay of the present invention to determine the presenceof prostaglandin D₂ synthase provides a simple means of differentiatingrenal wasting syndrome from SIADH. The bioassay of the present inventionalso provides simple means of differentiating Alzheimer's disease frommulti-infarct dementia. These clinical differentiations are oftendifficult to make. The importance of making a differentiation betweenboth renal salt wasting syndrome and SIADH is the difference intreatment modalities.

[0028] Clinical differentiation between Alzheimer's disease and otherdementia type of diseases such as multi-infarct dementia is also veryimportant particularly at the earliest stages of the disease whendiagnosis is very difficult. Early diagnosis of Alzheimer's disease maybe particularly helpful because it might lead to early treatment beforemore damage is done to the brain.

[0029] Diagnosis of Alzheimer's Disease and Renal Salt Wasting Syndromeby Detecting Prostaglandin D₂ Synthase

[0030] A sample is normally taken from a subject suspected of havingrenal salt wasting syndrome or Alzheimer's disease. This sample is thentested to measure the level of prostaglandin D₂ synthase. An elevatedlevel of prostaglandin D₂ synthase over control samples (e.g. one or twostandard deviations above normal, and especially levels more than twicethe normal level) is an indication of renal salt wasting syndrome or ofAlzheimer's disease. The method of the invention and detection kits inaccordance with the invention, preferably include comparison standardsderived from previously tested control samples. The method of theinvention may be practiced by comparing measured levels of prostaglandinD₂ synthase (in a test sample) to the comparison standards. Likelihoodthat the patient suffers from Alzheimer's disease or renal salt wastingsyndrome derived from correlation of measured levels to the comparisonstandards. The comparison standards may be any well known in the art,e.g. color change, phosphorescence, enzymatic activity or any otherparameter common in the art. Some examples are set forth below in thesection entitled “Methods of Detection of Prostaglandin D2 Synthase”.Naturally, the comparison standards should reflect control levelsmeasured by the same measurement technique as will be utilized formeasuring prostaglandin D₂ synthase in the patient sample. It ispreferred that the comparison standard show any age and gender-basedvariations.

[0031] Preferably, the samples to be tested are body fluids such asblood, plasma, urine, tears, saliva and the like. Both medical andveterinary applications are contemplated. In addition to human samples,samples may be taken from other mammals such as non-human primates,horses, swine, etc. In some instances it may be possible or evendesirable to dilute the sample prior to testing. Plasma, when used asthe sample, may be diluted, for example, with one or more fluidsselected from the group consisting of phosphate-buffered saline, pH7.0-7.4 (hereinafter “PBS”), PBS-containing TWEEN 20 (hereinafter “PBST”), PBS T with thimerosal (hereinafter “PBS TT”), PBS TT (gelatin)(hereinafter “PBS TTG”).

[0032] Preferred diluents and dilution ratios may vary in a known manneraccording to the sample being tested. In some instances, it can bedesirable to concentrate a sample that is initially too dilute. Prior totesting a sample whose pH is outside of the preferred pH for antibodyfunction (e.g. urine), the pH of the sample is preferably adjusted tobetween about 7.0 and 7.4, the preferred pH for antibody function.

[0033] Prostaglandin D₂ Antibody Preparation

[0034] (i) Polyclonal Antibodies

[0035] Polyclonal antibodies to prostaglandin D₂ or prostaglandin D₂fragments can generally be raised in animals by multiple subcutaneous(sc), intradermal (id), or intraperitoneal (ip) injections of natural orrecombinant prostaglandin D₂ synthase or prostaglandin D₂ synthasefragment or synthetic peptide and an adjuvant. It may be useful toconjugate prostaglandin D₂ synthase or a fragment containing the targetamino acid sequence to a protein that is immunogenic in the species tobe immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovinethyroglobulin, or soybean trypsin inhibitor using a bifunctional orderivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester(conjugation through cysteine residues), N-hydroxysuccinimide (throughlysine residues), glutaraldehyde, succinic anhydride, or SOCl2.

[0036] Animals can be immunized against the prostaglandin D₂ synthaseprotein or a fragment thereof, immunogenic conjugates, or derivatives bycombining 1 mg or 1 μg of the peptide or conjugate (for rabbits or mice,respectively) with 3 volumes of Freund's complete adjuvant or otheradjuvant and injecting the solution intradermally at multiple sites.Four to five weeks later the animals are boosted with ⅕ to {fraction(1/10)} the original amount of peptide or conjugate in Freund's completeadjuvant by subcutaneous injection at multiple sites or intradermalinjection at multiple sites of an equivalent amount of natural orrecombinant prostaglandin D₂ synthase. Seven to 14 days later theanimals are bled and the serum is assayed for prostaglandin D₂ synthaseor prostaglandin D₂ synthase fragment antibody titer. Animals areboosted until the titer plateaus. Preferably, the animal is boosted withpurified natural or recombinant prostaglandin D₂ synthase, the conjugateof the same prostaglandin D₂ synthase or prostaglandin D₂ synthasefragment, but conjugated to a different protein and/or through adifferent cross-linking reagent. Conjugates also can be made inrecombinant cell culture as protein fusions Also, aggregating agentssuch as alum may be used to enhance the immune response.

[0037] (ii) Monoclonal Antibodies

[0038] Monoclonal antibodies are obtained from a population ofsubstantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations that may be present in minor amounts. Thus, themodifier “monoclonal” indicates the character of the antibody as notbeing a mixture of discrete antibodies. For example, the prostaglandinD₂ synthase monoclonal antibodies of the invention may be made using thehybridoma method (Nature, 256: 495 (1975), or may be made by knownrecombinant DNA methods.

[0039] In the hybridoma method, a mouse or other appropriate hostanimal, such as a hamster, is immunized as hereinabove described toelicit lymphocytes that produce or are capable of producing antibodiesthat will specifically bind to the prostaglandin D₂ synthase orprostaglandin D₂ synthase fragment used for immunization. Alternatively,lymphocytes may be immunized in vitro. Lymphocytes then are fused withmyeloma cells using a suitable fusing agent, such as polyethyleneglycol, to form a hybridoma cell (Goding, Monoclonal Antibodies:Principles and Practice, pp.59-103 [Academic Press, 1986]).

[0040] The hybridoma cells thus prepared are seeded and grown in asuitable culture medium that preferably contains one or more substancesthat inhibit the growth or survival of the unfused, parental myelomacells. For example, if the parental myeloma cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (HAT medium), which substances prevent thegrowth of HGPRT-deficient cells.

[0041] Preferred myeloma cells are those that fuse efficiently, supportstable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Among these, preferred myeloma cell lines are murine myelomalines, such as those derived from MOPC-21 and MPC-11 mouse tumorsavailable from the Salk Institute Cell Distribution Center, San Diego,Calif. U.S.A., and SP-2 cells available from the American Type CultureCollection, Rockville, Md. U.S.A.

[0042] Culture medium in which hybridoma cells are growing is assayedfor production of monoclonal antibodies directed against prostaglandinD₂ synthase. Preferably, the binding specificity of monoclonalantibodies produced by hybridoma cells is determined byimmunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).

[0043] The binding affinity of the monoclonal antibody can, for example,be determined by the Scatchard analysis of Munson and Pollard, Anal.Biochem., 107: 220 (1980).

[0044] After hybridoma cells are identified that produce antibodies ofthe desired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods.Suitable culture media for this purpose include, for example, D-MEM orRPMI-1640 medium. In addition, the hybridoma cells may be grown in vivoas ascites tumors in an animal.

[0045] The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxyapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

[0046] DNA encoding the monoclonal antibodies of the invention isreadily isolated and sequenced using conventional procedures (e.g., byusing oligonucleotide probes that are capable of binding specifically togenes encoding the heavy and light chains of murine antibodies). Onceisolated, the DNA may be placed into expression vectors. Host cells arethen transformed or transfected with said vectors. Suitable host cellsinclude but are not limited to E. coli cells, simian COS cells, Chinesehamster ovary (CHO) cells, or myeloma cells that do not otherwiseproduce immunoglobulin protein, to obtain the synthesis of monoclonalantibodies in the recombinant host cells. Review articles on recombinantexpression in bacteria of DNA encoding an antibody include Skerra etal., Curr. Opinion in Immunol., 5: 256-262 (1993) and Pluckthun,Immunol. Revs., 130: 151-188 (1992).

[0047] The DNA also may be modified, for example, by substituting thecoding sequence for human heavy- and light-chain constant domains inplace of the homologous murine sequences (Morrison, et al., Proc. Nat.Acad. Sci., 81: 6851 [1984]), or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. In that manner, “chimeric” or “hybrid”antibodies are prepared that have the binding specificity of ananti-prostaglandin D₂ synthase monoclonal antibody herein.

[0048] Typically such non-immunoglobulin polypeptides are substitutedfor the constant domains of an antibody of the invention, or they aresubstituted for the variable domains of one antigen-combining site of anantibody of the invention to create a chimeric bivalent antibodycomprising one antigen-combining site having specificity for aprostaglandin D₂ synthase and another antigen-combining site havingspecificity for a different antigen.

[0049] Chimeric or hybrid antibodies also may be prepared in vitro usingknown methods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide-exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

[0050] (iii) Human Antibodies

[0051] Human monoclonal antibodies can be made by the hybridoma method.Human myeloma and mouse-human heteromyeloma cell lines for theproduction of human monoclonal antibodies have been described, forexample, by Kozbor, J. Immunol. 133, 3001 (1984); Brodeur, et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51-63(Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol.,147: 86-95 (1991).

[0052] It is now possible to produce transgenic animals (e.g. mice) thatare capable, upon immunization, of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy-chain joining region (J[H]) gene in chimeric andgerm-line mutant mice results in complete inhibition of endogenousantibody production. Transfer of the human germ-line immunoglobulin genearray in such germ-line mutant mice will result in the production ofhuman antibodies upon antigen challenge. See, e.g., Jakobovits et al.,Proc. Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature,362: 255-258 (1993); Bruggermann et al., Year in Immuno., 7: 33 (1993).

[0053] Alternatively, phage display technology (McCafferty et al.,Nature, 348: 552-553 [1990]) can be used to produce human antibodies andantibody fragments in vitro, from immunoglobulin variable (V) domaingene repertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B-cell. Phage display can be performed in avariety of formats; for their review see, e.g., Johnson, Kevin S. andChiswell, David J., Current Opinion in Structural Biology, 3: 564-571(1993). Several sources of V-gene segments can be used for phagedisplay. Clackson et al., Nature, 352: 624-628 (1991) isolated a diversearray of anti-oxazolone antibodies from a small random combinatoriallibrary of V genes derived from the spleens of immunized mice. Arepertoire of V genes from unimmunized human donors can be constructedand antibodies to a diverse array of antigens (including self-antigens)can be isolated essentially following the techniques described by Markset al., J. Mol. Biol., 222: 581-597 (1991), or Griffith et al., EMBO J.,12: 725-734 (1993).

[0054] In a natural immune response, antibody genes accumulate mutationsat a high rate (somatic hypermutation). Some of the changes introducedwill confer higher affinity, and B cells displaying high-affinitysurface immunoglobulin are preferentially replicated and differentiatedduring subsequent antigen challenge. This natural process can bemimicked by employing the technique known as “chain shuffling” (Marks etal., Bio/Technol., 10: 779-783 [1992]). In this method, the affinity of“primary” human antibodies obtained by phage display can be improved bysequentially replacing the heavy and light chain V region genes withrepertoires of naturally occurring variants (repertoires) of V domaingenes obtained from unimmunized donors. This technique allows theproduction of antibodies and antibody fragments with affinities in thenM range. A strategy for making very large phage antibody repertoireshas been described by Waterhouse et al., Nucl. Acids Res., 21: 2265-2266(1993).

[0055] Gene shuffling can also be used to derive human antibodies fromrodent antibodies, where the human antibody has similar affinities andspecificities to the starting rodent antibody. According to this method,which is also referred to as “epitope imprinting”, the heavy or lightchain V domain gene of rodent antibodies obtained by phage displaytechnique is replaced with a repertoire of human V domain genes,creating rodent-human chimeras. Selection on antigen results inisolation of human variable capable of restoring a functionalantigen-binding site, i.e. the epitope governs (imprints) the choice ofpartner. When the process is repeated in order to replace the remainingrodent V domain, a human antibody is obtained (see PCT WO 93/06213,published 1 Apr. 1993). Unlike traditional humanization of rodentantibodies by CDR grafting, this technique provides completely humanantibodies, which have no framework or CDR residues of rodent origin.

[0056] Methods of Detection of Prostaglandin D₂ Synthase

[0057] Detection with Antibodies

[0058] For diagnostic applications (i.e. detection of prostaglandin D₂synthase), antibodies against prostaglandin D₂ synthase typically willbe labeled with a detectable moiety. The detectable moiety can be anyone which is capable of producing, either directly or indirectly, adetectable signal. For example, the detectable moiety may be aradioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I; a fluorescent orchemiluminescent compound (Melegos et al., Clin. Chem. 42:12 (1996)),such as fluorescein isothiocyanate, rhodamine, or luciferin; radioactiveisotopic labels, such as, e.g., ¹²⁵i, ³²P, ¹⁴C, or ³H; or an enzyme,such as alkaline phosphatase, beta-galactosidase, or horseradishperoxidase.

[0059] Any method known in the art for separately conjugating theantibody to the detectable moiety may be employed, including thosemethods described by Hunter et al., Nature, 144: 945 (1962); David etal., Biochemistry, 13: 1014 (1974); Pain et al., J. Immunol. Meth., 40:219 (1981); and Nygren, J. Histochem, and Cytochem., 30: 407 (1982).

[0060] The antibodies used for diagnostic purposes in the presentinvention may be employed in any known assay method, such as competitivebinding assays, direct and indirect sandwich assays, andimmunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual ofTechniques, pp. 147-158 (CRC Press, Inc., 1987).

[0061] Competitive binding assays rely on the ability of a labeledstandard (which may be prostaglandin D₂ synthase or an immunologicallyreactive portion thereof) to compete with the test sample for bindingwith a limited amount of antibody. The amount of prostaglandin D₂synthase in the test sample is inversely proportional to the amount ofstandard that becomes bound to the antibodies. To facilitate determiningthe amount of standard that becomes bound, the antibodies generally areinsolubilized before or after the competition, so that the standard andprostaglandin D₂ synthase from the tested sample that are bound to theantibodies may conveniently be separated from the unbound material.

[0062] Sandwich assays involve the use of two antibodies, each capableof binding to a different immunogenic portion, or epitope, of theprotein (prostaglandin D₂ synthase) to be detected. In a sandwich assay,the test sample protein (prostaglandin D₂ synthase) is bound by a firstantibody which is immobilized on a solid support, and thereafter asecond antibody binds to the protein, thus forming an insolublethree-part complex. David and Greene, U.S. Pat. No. 4,376,110. Thesecond antibody may itself be labeled with a detectable moiety (directsandwich assays) or may be measured using an anti-immunoglobulinantibody that is labeled with a detectable moiety (indirect sandwichassay). For example, one type of sandwich assay is an ELISA assay(Enzyme Linked immunoabsorbent assay), in which case the detectablemoiety is an enzyme (e.g., horseradish peroxidase).

[0063] Prostaglandin D₂ synthase antibodies are useful in diagnosticassays for prostaglandin D₂ synthase, e.g., its production in specificcells or tissues, or its presence in urine or serum. The antibodies arelabeled and/or are immobilized on an insoluble matrix. In oneembodiment, an antibody that binds to prostaglandin D₂ synthase isimmobilized on an insoluble matrix, the test sample is contacted withthe immobilized antibody composition to adsorb all prostaglandin D₂synthase, and then the immobilized prostaglandin D₂ synthase moleculesare contacted with antibodies that recognize different antigenic siteson prostaglandin D₂ synthase, these antibodies being identifiable by aunique label such as discrete fluorophores or the like. By determiningthe presence and/or amount of the unique label, the amount ofprostaglandin D₂ synthase can be determined.

[0064] Competitive assays rely on the ability of a tracer (i.e.labelled) analogue to compete with the test sample prostaglandin D₂synthase for a limited number of binding sites on a common bindingpartner. The binding partner generally is insolubilized before or afterthe competition and then the tracer and prostaglandin D₂ synthase boundto the binding partner are separated from the unbound tracer andprostaglandin D₂ synthase. This separation is accomplished by decanting(where the binding partner was preinsolubilized) or by centrifuging(where the binding partner was precipitated after the competitivereaction). The amount of test sample prostaglandin D₂ synthase isinversely proportional to the amount of bound tracer as measured by theamount of marker substance. Dose-response curves with known amounts ofprostaglandin D₂ synthase are prepared and compared with the testresults to quantitatively determine the amount of prostaglandin D₂synthase present in the test sample. These assays are called ELISAsystems when enzymes are used as the detectable markers.

[0065] Another species of competitive assay, called a “homogeneous”assay, does not require a phase separation. Here, a conjugate of anenzyme with prostaglandin D₂ synthase is prepared and used such thatwhen anti-prostaglandin D₂ synthase binds to the prostaglandin D2synthase, the presence of the anti-prostaglandin D₂ synthase modifiesthe enzyme activity. In this case, prostaglandin D₂ synthase or itsimmunologically active fragments are conjugated with a bifunctionalorganic bridge to an enzyme such as peroxidase. Conjugates are selectedfor use with anti-prostaglandin D₂ synthase so that binding of theanti-prostaglandin D₂ synthase inhibits or potentiates the enzymeactivity of the label. This method per se is widely practiced under thename of EMIT.

[0066] Sandwich assays particularly are useful for the determination ofprostaglandin D₂ synthase. In sequential sandwich assays an immobilizedbinding partner is used to adsorb test sample prostaglandin D₂ synthase,the test sample is removed as by washing, the bound prostaglandin D₂synthase is used to adsorb labeled binding partner, and bound materialis then separated from residual tracer. The amount of bound tracer isdirectly proportional to test sample prostaglandin D₂ synthase. In“simultaneous” sandwich assays the test sample is not separated beforeadding the labeled binding partner. A sequential sandwich assay using ananti-prostaglandin D₂ synthase monoclonal antibody as one antibody and apolyclonal anti-prostaglandin D₂ synthase antibody as the other isuseful in testing samples for prostaglandin D₂ synthase presence.

[0067] Detection with Assay for Apoptosis

[0068] Applicant demonstrated the presence of a factor (isolated andidentified as prostaglandin D₂ synthase) in the plasma of patients withAlzheimer's disease that increases apoptosis in cultured LLC-PK1 cellswhen compared to plasma from control subjects (C) and subjects sufferingfrom multi-infarct dementia (MID). To verify this result, applicant alsodemonstrated (infra) that addition of -Δ12prostaglandin J₂ to culturedLLC-PK1 cells increases the rate of apoptosis.

[0069] The determination of apoptosis can be done in a variety of wayssuch as a TUNEL assay, demonstration of a nucleosomal ladder by agarosegel electrophoresis, and electron micrographic analysis showing typicalmorphology of apoptosis.

[0070] The conclusion that apoptosis results from the presence of afactor (i.e. prostaglandin D₂ synthase) in the plasma of patientssuffering from Alzheimer's disease is based on observed DNA degradationin nuclei of affected cells. The degree of apoptosis was dose and timedependent, continually increasing up to at least 8 h with renewedsampling of Alzheimer's plasma every 2 h. The apoptotic ladder seen byagarose gel electrophoresis results from the double-strandedendonucleolytic cleavage of DNA which occurs at the linker regions ofnucleosomes to produce fragments of multiples of about 180 bp. Thisfragmentation of DNA appears coincident with condensation of nuclearchromatin prior to cell death and is considered a characteristicbiochemical feature of apoptosis (Y. Gavrieli et al., J. Cell. Biol.119:493-501, 1992). Demonstration of this repeat pattern was, therefore,used as an indicator of apoptosis. The 3′OH ends of this cleaved DNA canalso serve as substrate for deoxynucleotidyl terminal transferase TdT,which led to the development of TdT-mediated dUTP-biotin nick endlabeling (TUNEL) (Y. Gavrieli et al., J. Cell. Biol. 119:493-501, 1992).This technique results in the labeling of nuclei in-situ, prior to theappearance of the ladder by gel electrophoresis. TUNEL staining of DNAfragments occurs not only in histologically-defined apoptotic cells butalso in intact cells during the early stages of apoptosis (Y. Gavrieliet al., J. Cell. Biol. 119:493-501, 1992). Electron microscopy of thenuclei of LLC-PK1 cells shows chromosomal fragmentation (nuclearcondensation) upon exposure to prostaglandin D₂ synthase.

[0071] Prostaglandin D₂ synthase may also be detected in an enzymaticassay according to the method described by Urade et al. (J. Bio. Chem.270: 1422-1428; 1995).

[0072] The foregoing are merely exemplary diagnostic assays fordetection of prostaglandin D₂ synthase in accordance with the invention.Because it is the level of prostaglandin D₂ synthase that is relevant,any other technique that effectively measures prostaglandin D₂ synthaseis also included within the scope hereof.

[0073] Treatment of Alzheimer's Disease and Renal Salt Wasting Syndrome

[0074] Prostaglandin D₂ synthase plays a role in the synthesis pathwayof Δ¹²prostaglandin J₂. Arachidonic acid is initially converted bycyclo-oxygenase to prostaglandin H₂. Prostaglandin D₂ synthase, is anenzyme that converts prostaglandin H₂ to prostaglandin D₂. ProstaglandinD₂ then spontaneously converts to Δ¹²prostaglandin J₂, presumably thebiologically active metabolite of this pathway.

[0075] Without intending to be bound by theory, it is believed that thepresence of prostaglandin D₂ synthase in the urine and blood of patientssuffering from Alzheimer's disease or renal salt wasting syndrome is anindication of an excess of this enzyme at least in some regions of thepatient's body which evidently results in excess production of-Δ¹²prostaglandin J₂. Along this line, more prostaglandin H₂ getsconverted into prostaglandin D₂ which is spontaneously (i.e. immediatelyand without the need of an enzyme) converted to prostaglandin J₂ andthen to -Δ¹²prostaglandin J₂.

[0076] Applicant found that prostaglandin D₂ synthase increasedapoptosis of human kidney proximal tubule cells in culture. However,-Δ¹²prostaglandin J₂ was the only prostaglandin in the above pathwaythat induces apoptosis. -Δ¹²Prostaglandin J₂ increased apoptosis to thesame degree as prostaglandin D₂ synthase. The addition of prostaglandinD₂ synthase and indomethacin, which inhibits cyclo-oxygenase and reducesthe prostaglandin synthesis downstream did not increase apoptosis abovebaseline. The inhibition of prostaglandin D₂ synthase by N-EthylMaleimide inhibited apoptosis. In addition, combination of indomethacin,prostaglandin D₂ synthase and -Δ¹²prostadlandin J₂ increased apoptosis.Furthermore, addition of -Δ¹²prostadlandin J₂ to indomethacin increasedapoptosis. All these results indicate that prostaglandin D₂ synthasecauses apoptosis by helping to produce more of -Δ12prostaglandin J₂.Thus, the present invention seeks to reduce -Δ¹²prostaglandin J₂ levels.

[0077] Furthermore, it is believed that Alzheimer's disease is theresult of neuronal brain cells undergoing apoptotic cell death. It isalso believed that renal salt wasting syndrome might be the result ofapoptotic cell death by kidney tubule cells. Therefore, inhibiting therate of apoptosis which, at least in part, is caused by elevated-Δ¹²prostaglandin J₂ levels is expected to be an effective treatment forboth Alzheimer's disease and renal salt wasting syndrome.

[0078] Accordingly, the present invention provides methods of (1)treating or reducing risk of onset of renal salt wasting syndrome, (2)inhibiting the rate of apoptosis, and (3) reducing the risk of onset, ortreating (e.g. by slowing the rate of progression of) Alzheimer'sdisease

[0079] The methods inhibit the effect of, or reduce the levels of-Δ¹²prostaglandin J₂ levels.

[0080] Reduction of -Δ¹²prostaglandin J₂ activity

[0081] The reduction of -Δ12prostaglandin J₂ levels can be accomplishedin a wide variety of ways, for example those set forth below.

[0082] 1) Inhibiting the rate of synthesis of -Δ¹²prostaglandin J₂. Thiscan be accomplished by administering at least one agent selected fromthe group consisting of cyclo-oxygenase inhibitor, cyclo-oxygenaseantibody, prostaglandin D₂ synthase inhibitor, prostaglandin D₂ synthaseantibody. The cyclo-oxygenase inhibitor can be for example indomethacinand prostaglandin D₂ synthase inhibitor can be N-ethyl maleimide.

[0083] 2) Increasing the rate of degradation or elimination of-Δ¹²prostaglandin J₂. This can be accomplished for example by adding anagent that increases the rate of catabolism or the rate of turnover of-Δ¹²prostaglandin J₂.

[0084] 3) Administering to the subject an inhibitor of -Δ¹²prostaglandinJ₂ (e.g. a receptor antagonist)

[0085] Pharmaceutical Administration

[0086] In accordance with one aspect of the invention, once Alzheimer'sdisease or renal salt wasting syndrome is diagnosed, at least one agentselected from the group of cyclo-oxygenase inhibitor, cyclo-oxygenaseantibody, prostaglandin D₂ synthase inhibitor, prostaglandin D₂ synthaseantibody, and Δ¹²prostaglandin J₂ inhibitor, is(are) administered at adosage sufficient to reach the affected location (for example, the brainor kidney) and reduce the rate of apoptosis. Non-limiting examples ofmethods of administration and dosages which apply to both treatment andprevention are detailed below. Dosages will be the same when theinvention is used prophylactically, preferably for patients at higherrisk than the general population of acquiring the disease in question.Risk factors are known in the art. As used herein, a “patient” may be ahuman or other mammalian patient. Veterinary use of the inventionsherein are appropriate.

[0087] As used in the invention, any of the above-identified agents maybe administered with or without additional carrier or diluent by theoral, systemic, percutaneous, transmucosal, or other typical route. In apharmaceutical composition for oral administration, an agent asdescribed above is preferably present in a concentration between 5 and99% by weight relative to total weight of the composition, morepreferably between 50 and 99 percent, especially between 80 and 99percent.

[0088] When prepared for percutaneous administration, an agent ispreferably present in a concentration between 2 and 20% by weightrelative to the total weight of the composition, more preferably between5 and 15%, especially between 5 and 10%.

[0089] Oral Administration

[0090] When administered by the oral route, the agent describedhereinabove may be formulated with conventional pharmaceuticalexcipients, e.g. spray dried lactose and magnesium stearate, intotablets or capsules for oral administration at concentrations providingeasy dosage in a range from 1 ng to 10 g, preferably, from 1-10 mg perday per kg of body weight.

[0091] The active substance can be worked into tablets or dragee coresby being mixed with solid, pulverulent carrier substances, such assodium citrate, calcium carbonate or dicalcium phosphate, and binderssuch as polyvinyl pyrrolidone, gelatin or cellulose derivatives,possibly by adding also lubricants such as magnesium stearate, sodiumlauryl sulfate, “Carbowax” or polyethylene glycol. Of course,taste-improving substances can be added in the case of oraladministration forms. The active substance can be also administered insolid dispersion state in appropriate carriers. Such carriers may, forexample, be chosen from the group consisting of polyethylene glycols ofmolecular weight varying from 1,000 to 20,000 daltons andpolyvinylpyrrolidone (e.g., Povidone from American Chemicals Ltd.,Montreal, Canada).

[0092] As further forms, one can use plug capsules, e.g. of hardgelatin, as well as closed soft-gelatin capsules comprising a softeneror plasticizer, e.g. glycerine. The plug capsules contain the activesubstance preferably in the form of granulate, e.g. in mixture withfillers, such as lactose, saccharose, mannitol, starches such as potatostarch or amylopectin, cellulose derivatives or highly dispersed silicicacids. In soft-gelatin capsules, the active substance is preferablydissolved or suspended in suitable liquids, such as vegetable oils orliquid polyethylene glycols.

[0093] Topical Administration

[0094] For the treatment of conditions associated with apoptosis of theskin, the preferred mode of administration is topical. Anypharmaceutically acceptable base typically used in the art for preparingformulations in the form of topical gels, ointments, lotions, or thelike may be used as the base. The agent described above is preferablyprovided at a concentration of 0.001-10%, more preferably 0.1-1% byweight of the total formulation. One to two applications per day to theaffected area are recommended.

[0095] Transdermal Delivery

[0096] When the composition of the present invention is formulated as anointment, lotion, gel, cream or the like, for transdermaladministration, the active compound is admixed with a suitable carrierwhich is compatible with human skin or mucosa and which enhancestransdermal or transmucosal penetration of the compound through the skinor mucosa. Suitable carriers are known in the art and include but arenot limited to Klucel HF and Glaxal base which is available from GlaxalCanada Limited. Other suitable vehicles can be found in Koller and Buri,S. T. P. Pharma 3(2), 115-124, 1987. The carrier is preferably one inwhich the active ingredient(s) is(are) soluble at ambient temperature atthe concentration of active ingredient that is used. The carrier shouldhave sufficient viscosity to maintain the precursor on a localized areaof skin or mucosa to which the composition has been applied, withoutrunning or evaporating for a time period sufficient to permitsubstantial penetration of the precursor through the localized area ofskin. The carrier is typically a mixture of several components, e.g.pharmaceutically acceptable solvents and a thickening agent. A mixtureof organic and inorganic solvents can aid hydrophilic and lipophilicsolubility, e.g. water and an alcohol such as ethanol. Desirably, thecarrier is one which, if applied twice daily in an amount providing 1 ngto 10 g, preferably 1 mg to 1 g, and more preferably 100 mg 1 g of agentto the afflicted area, will provide blood serum levels sufficient toreduce apoptosis in the effected tissues.

[0097] The carrier may include various additives commonly used inointments, lotions, gels, and creams and well known in the cosmetic andmedical arts. For example, fragrances, antioxidants, perfumes, gellingagents, thickening agents such as carboxymethylcellulose, surfactants,stabilizers, emollients, coloring agents and other similar agents may bepresent.

[0098] The lotion, ointment, gel or cream should be thoroughly rubbedinto the skin so that no excess is plainly visible, and the skin wouldnot be washed in that region until most of the transdermal penetrationhas occurred, preferably, at least 15 minutes and, more preferably, atleast 30 minutes after application.

[0099] A transdermal patch may be used to deliver the composition of thepresent invention in accordance with known techniques. It is typicallyapplied for a long period, e.g. 0.5 to 4 days, but typically contactsactive ingredients to a smaller surface area, allowing a slow andconstant delivery of active ingredient.

[0100] A number of transdermal drug delivery systems that have beendeveloped, and are in use, are suitable for delivering the activeingredient of the present invention. The rate of release is typicallycontrolled by a matrix diffusion, or by passage of the active ingredientthrough a controlling membrane.

[0101] Mechanical aspects of transdermal devices are well known in theart, and are explained, for example, in U.S. Pat. Nos. 4, 162,037,5,154,922, 5,135,480, 4,666,441, 4,624,665, 3,742,951, 3,797,444,4,568,343, 4,064,654, 5,071,644, 5,071,657, the disclosures of which areincorporated herein by reference. Additional background is provided byEuropean Patent 0279982 and British Patent Application 2185187.

[0102] The device may be any of the general types known in the artincluding adhesive matrix and reservoir-type transdermal deliverydevices. The device may include drug-containing matrixes incorporatingfibers which absorb the active ingredient and/or carrier. In areservoir-type device, the reservoir may be defined by a polymermembrane impermeable to the carrier and to the active ingredient.

[0103] In a transdermal device, the device itself maintains activeingredient in contact with the desired localized skin surface. In such adevice, the viscosity of the carrier for active ingredient is of lessconcern than with a cream or gel. A solvent system for a transdermaldevice may include, for example, oleic acid, linear alcohol lactate anddipropylene glycol, or other solvent systems known in the art. Theactive ingredient may be dissolved or suspended in the carrier.

[0104] For attachment to the skin, a transdermal patch may be mounted ona surgical adhesive tape having a hole punched in the middle. Theadhesive is preferably covered by a release liner to protect it prior touse. Typical material suitable for release includes polyethylene andpolyethylene-coated paper, and preferably silicone-coated for ease ofremoval. For applying the device, the release liner is simply peeledaway and the adhesive attached to he patient's skin. In U.S. Pat. No.4,135,480, the disclosure of which is incorporated by reference, Bannonet al. described an alternative device having a non-adhesive means forsecuring the device to the skin.

[0105] Intravenous Injection

[0106] Sterile solutions can also be administered intravenously. Theactive ingredient may be prepared at a final dose of 1 ng to 10 g,preferably 1 mg to 1 g per Kg of body weight as a sterile solidcomposition which may be dissolved or suspended at the time ofadministration using sterile water, saline, or other appropriate sterileinjectable medium. Carriers are intended to include necessary and inertbinders, suspending agents, lubricants, flavorants, sweeteners,preservatives, dyes, and coatings.

[0107] Preferred Uses of the Invention

[0108] The invention is applicable to both diagnostic, prevention andtreatment purposes. A non-exclusive list of diagnostic uses is set forthin column 1 of Table 1 below. Columns 2-4 set forth, for each use,preferences regarding the manner in which certain diagnostic tests maybe varied for best results. TABLE 1 Preferred Diagnostic test/Detectionof Preferred Preferred Method of Prostaglandin D₂ synthase PopulationSample Detection Renal Salt Wasting General, Urine or ELISA Syndromeespecially Plasma symptomatic patients Alzheimer’s Disease Patients withUrine or ELISA dementia Plasma

[0109] A non-exclusive list of treatment uses is set forth in column 1of Table 2 below. Columns 2-4 set forth, for each use, preferencesregarding the preferred pharmaceutical agent (s) to be used, the dosageand the manner of administration. TABLE 2 Pharmaceutical Treatment AgentDosage Administration Renal Salt Wasting 1) Cyclo-oxygenase  50 mg Oral,three times Syndrome inhibitors such as daily indomethacin 2)Prostaglandin D₂ 240 mg Intravenous, once synthase daily monoclonalantibodies Alzheimer's Disease 1) Prostaglandin D₂ 240 mg Intravenous,once synthase daily monoclonal antibodies

[0110] Experimental Details

[0111] Patient Selection. Patients were randomly recruited at theDivision of Geriatric psychiatry, UMDNJ-Robert Wood Johnson MedicalSchool based on their willingness to participate in the study. Allsubjects were examined by a board certified geriatric neuropsychiatristwho established the diagnosis of dementia. The bioassay was performed atWinthrop University Hospital. The protocol for these studies wasapproved by the respective institutional review boards of bothinstitutions. Consent from demented patients was obtained from theirlegal guardian on all cases. Seventeen subjects with Alzheimer's diseasemet NINCDS-ADRDA criteria for probable Alzheimer 's disease (G. McKhannet al., Neurol. 34:939-944, 1984) and 11 multi-infarct dementia (MID)subjects met DSM-IIIR criteria for the diagnosis of MID and hadHachinski Ichemia Scale scores greater than 7 (American PsychiatricAssociation. Diagnostic and Statistical manual of mental disorder. 4thedition (1994). Am. Psychiatric Assoc. Washington, D.c. Dementia WorkGroup: Gary J. Tucker, Chairperson; V.C. Hachinski et al., Arch. Neurol.32:632-637, 1975). Nine subjects of the same age and gender distributionserved as normal controls (C). In addition to the routine testing, allpatients received either a CT scan or magnetic resonance imaging ofbrain and Mini-Mental State Examination (MMSE) score (M.F. Folstein etal., J. Psychiatric Res. 12:189-198, 1975). Heparinized whole blood fromall subjects was centrifuged at 1500 g for 10 minutes at 4° C. within 30min. after collection; the plasma was then transferred to a new plastictube and stored at −70° C. all samples were stored at −70° C. until timeof bioassay, except during overnight shipping on dry ice.

[0112] Cell Culture and Assay Protocol. LLC-PK1, a pig kidney epithelialcell line was plated at a density of 10³ cells per well into eight-wellPermanox plastic chamber slides (NUNC, Naperville, Ill.). The cells werecultured at 37° C. in 5% CO₂ in humidified incubators and grown for 3days to 70-80% confluency in DMEM-F12 that was supplemented with 10%fetal calf serum, 7.5% sodium bicarbonate, 15 mM HEPES, 200 mML-Glutamate, 100 u penicillin and 0.1 ug/ml streptomycin (LifeTechnologies, Gaithersburg, MD). The culture fluid was then removed andcells were exposed to plasma from control individuals, Alzheimer'sdisease patients or multi-infarct dementia patients diluted 1:5 in freshDMEM-F12 media, supplemented as above, for 2 h at 37° C. The cells werethen rinsed in PBS, and fixed in 4% formaldehyde in PBS for 10 min.,permeabilized with 0.5% Triton X-100 (Sigma Chemical CO., St. Louis,Mo.) for 5 min. and washed in 4 changes of distilled water. A positivecontrol was obtained by exposing cells to 0.6 mM H₂O₂ diluted inDMEM-F12 for 2 h.

[0113] Apoptosis Assay (TUNEL): Nuclear DNA fragmentation consistentwith apoptosis was determined by the method of TdT-mediated dUTP-biotinnick-end labeling (TUNEL) (Y. Gavrieli et al., J. Cell. Biol.119:493-501, 1992). The ApopDetek cell death assay kit (Enzo,Farmingdale, N.Y.) was used utilizing terminal deoxynucleotidetransferase to incorporate Bio-16-dUTP onto the 3′-OH termini in the DNAof apoptotic cells, subsequent binding with streptavidin-horseradishperoxidase, and visualization after conversion of the substrate andchromagen (hydrogen peroxide and aminoethylcarbazole) into a localizedbrick red precipitate. A blue counter stain was also used. Slides werethen observed for morphologically irregular and condensed nuclei whichcontain dark red precipitate to indicate TUNEL-positive cells using aNikon (Nikon, Inc., Melville, N.Y.) Optiphot microscope. Five to sixrandom field totaling approximately 1,000 to 1,500 cells were countedper slide. Apoptotic index (AI), defined as the percent of cellsundergoing apoptosis, is calculated by dividing the number of positivenuclei by the total number of nuclei counted multiplied by 100.

[0114] Dose and Time-Response Studies. The TUNEL assay was performed inLLC-PK1 cells that were exposed to different dilutions of plasma ofpatients with Alzheimer's disease and control plasma at differentintervals of time. Alzheimer's disease and control plasma were dilutedwith DMEM-F12 at 1:100, 1:20, 1:10; 1:5, 1:3 and 1:2 and added to 70-80%confluent LLC-PK1 cells for 2 h; conversely, Alzheimer's disease andcontrol plasma were diluted 1:5 with DMEM-F12 and exposed to LLC-PK1cells for 60, 90, 120 and 180 min. The selection of 2 h exposure in thedilution studies and 1:5 dilution of plasma in the time response studieswere based on maximum apoptotic index noted with the respective studies.

[0115] Electron Microscopy. LLC-PK1 cells were plated at 10³ cells per35 mm plastic petri dish, exposed to Alzheimer's disease or controlplasma at 1:5 dilution in DMEM-F12 for 2 h and fixed with 2.5%glutaraldehyde in 0.1M sodium cacodylate, pH 7.2, for 1 h at 4° C. Thecells were then postfixed in 1% buffered osmium tetroxide, dehydrated ina graded series of ethanol, and embedded in LX112 (Ladd ResearchIndustries, Burlington, VT.). En fac and cross-sectional thin sectionswere stained with uranyl acetate and lead citrate and examined on aZeiss EM10 transmission electron microscope.

[0116] DNA Ladder Assay. DNA ladder was observed using a modification ofthe procedure described by Eastman (Eastman, A. “Assays for FeaturesAssociated with Apoptosis” in Meth. Cell Biol. 46:41-55 edited by L. M.Schwartz and B. A. Osborne, Academic Press). LLC-PK1 cells (10⁶) wereseeded into T75 flasks (Falcon) containing 10 ml of DMEM-F12-10% fetalcalf serum supplemented with 0.12% NaHCO₃, 5 mM glutamine, 15 mM HEPESand 1% pen/strep. Cells were allowed to attach overnight at 37° C. in 5%humidified CO₂. Five ml of medium were withdrawn and 0.5 ml of testplasma added. Cells in the medium and adherent cells (0.05% trypsin in0.53 mM EDTA, GIBCO, 3 min., 37° C.) were harvested on days 2, 3, 4, and5 by centrifugation at 142×g for 3 min. at room temperature. Thecellpellet was warmed to 50° C. for 2-3 min. and resuspended in 2% SeaPlaque agarose (FMC, Rockland, Me.) in 0.125 M EDTA pH 7.4, anddispensed into a precooled (4° C.) mold. The agarose plugs wereincubated at 50° C. for 2 h in 0.5 M EDTA pH 8.0, 1% sarcosine (Sigma),and 1 mg/ml of proteinase K (Boehringer Mannheim). Plugs were thenincubated at 37° C. for 30 min. in 10×volume of 10 mM TrisHCl pH 7.5, 50mM EDTA. The buffer was exchanged with TE (10 mM TrisHCl pH 7.5, 1 mMEDTA), RNase A, previously boiled for 15 min (Sambrook, J., E. F.Fritsch, and T. Maniatis, “Molecular Cloning: A Laboratory Manual”, ColdSpring Harbor, N.Y., Cold Spring Harbor Laboratory Press, 1989), wasadded to a final concentration of 250 μg/ml, and the plugs incubated anadditional 50 min. at 37° C. DNA in the plugs was subjected toelectrophoresis through a 2% SeaKem (FMC) agarose gel in TAE buffer(Sambrook, J., E. F. Fritsch, and T. Maniatis, “Molecular Cloning: ALaboratory Manual”, Cold Spring Harbor, N.Y., Cold Spring HarborLaboratory Press, 1989) at 2V/cm for 14 h and visualized as described(Eastman, A. “Assays for Features Associated with Apoptosis” in Meth.Cell Bio. 46:41-55 edited by L. M. Schwartz and B. A. Osborne, AcademicPress).

[0117] Partial Protein Purification and Heating of Plasma. Pooled plasmafrom Alzheimer's disease and control subjects were dialyzed in a 10 kDam.w. cut-off membrane in 20 mM phosphate buffer, pH 7.1, and centrifugedat 13,000 g for 15 min. The clear supernatant was loaded into 10 ml ofan Affi-Gel Blue Gel affinity column (Bio-Rad Laboratories, Hercules,Calif.). The column was then washed with loading buffer until proteinlevels were not detectable, followed by the sequential elution with 0.5M and 2 M NaCl in buffer. Protein concentration was monitored by UVabsorbance at 280 nm. The three protein fractions (load and wash, 0.5 MNaCl and 2 M NaCl) were dialyzed in a 10 kDa m.w. cut-off membrane,concentrated over a bed of PEG 8000 in a 1 kDa m.w. cut-off membrane anddialyzed in a 10 kDa m.w. cut-off membrane against 10 mM phosphatebuffer, pH 7.1. Cultured LLC-PK1 cells were then exposed to 30-100 ug ofthe two pooled fractions for 2 h at 37° C. and a TUNEL assay performed.

[0118] In separate experiments, Alzheimer's disease and control plasmawere heat-treated at 56° C. for 30 min. In some experiments, plasma wasboiled at 100° C. for 5 min. and the denatured protein aggregatesremoved by sedimentation at 1,000 g for 1 min. prior to testing by TUNELassay as noted above. In separate experiments, Alzheimer's diseaseplasma was alternately frozen at −70° C. and thawed to room temperatureat least 3 times and a TUNEL assay performed in LLC-PK1 cells after a 2h exposure to a 1:5 dilution of the plasma with DMEM-F12 at 37° C.

[0119] Isoelectric Focusing. The active fraction from the Affi-GelBlue-Gel run (0.5 M NaCl) was further fractionated by isoelectricfocusing (IEF) using Fotofor (Bio-Rad). This active fraction was run ata pH gradient of 3-10, using Bio-Lyte ampholyte {fraction (3/10)}, at aconstant power of 15 W at 4° C. for 4 h. Fractions were pooled accordingto their protein profile and assayed for apoptotic activity. Fractionswith highest apoptotic index were pooled, dialyzed and refractionated byIEF using a narrow pH gradient of 4-6, at the same settings, utilizingBio-Lyte ampholyte {fraction (4/6)} and {fraction (3/10)}, 80:20%,respectively (Bio-Rad Laboratories, Hercules, Calif.).

[0120] Effect of Protein Synthesis on Apoptotic Activity. LLC-PK1 cellswere exposed to Alzheimer's disease and control plasma in the absenceand presence of cycloheximide (0.2-200 uM) (Sigma, St. Louis, Mo.).Apoptotic index was measured in these cells by TUNEL assay.

[0121] Effect of Calcium Depletion on Factor Activity. The TUNEL assaywas performed in the usual manner except for substituting DMEM-F12 withcalcium-free DMEM, supplemented with dialyzed 10% fetal calf serum (LifeTechnologies, Grand Island, N.Y.) and 0.6 mM EGTA to chelate calcium.Dialyzed Alzheimer's disease and control plasma were then added to thecalcium-free media for 2 h at 37° C. and a TUNEL assay performed.

[0122] Contribution of Known Apoptotic Inducers. To test the possibilitythat the apoptotic factor in Alzheimer's disease plasma was β amyloid,TNF-α or myeloma light chain, the TUNEL assay was repeated as aboveafter a 2 h incubation at 37° C. with 0.10-50 mM β amyloid (PeninsulaLaboratories, Inc., Belmont, Calif.), 5 pM-3 nM TNF-α (Quantikine,Minneapolis, Minn.) and 3-60 ug X myeloma light chain, kindly suppliedby Dr. Vecihi Batuman, Tulane University School of Medicine, NewOrleans, La. To eliminate the possibility that a protease in Alzheimer'sdisease plasma is responsible for the apoptotic activity, the effect ofa broad spectrum protease inhibitor cocktail (Boehringer Mannheim) wasstudied on apoptotic index using the TUNEL assay. LCC-PK1 cells wereincubated with Alzheimer's disease plasma with and without the inhibitorcocktail and assayed as detailed above. This cocktail inhibits a largespectrum of serine, cysteine, and metalloproteases as well as calpains.It consists of aprotinin, leupeptin, EDTA, and pefabloc.

[0123] Statistical Analysis. All TUNEL assays were performed intriplicate and the data expressed as the mean±SEM. An unpaired Student'sT test was used to compare one set of experiments from the other and aP<0.05 was deemed significant. A multivariate analysis was used todetermine whether any medications taken by the patients might affect theresults or if there was a correlation between apoptotic index and MMSEscore.

EXAMPLE 1 A Factor in the Plasma of Patients with Alzheimer's DiseaseCauses Apoptosis; Partial Purification and Characterization of theFactor

[0124] Electron microscopy of LLC-PK1 cells after incubation withAlzheimer's disease plasma (see Experimental Details) illustrates thedistinct pattern of apoptotic cells. Apoptotic cells have condensed,black nuclei and some cells are noted to be shrunken and engulfed byneighboring cells, FIG. 1. Light microscopic view of these cells thathad been labeled in situ by TUNEL method are depicted in FIG. 2.

[0125] Table 3 summarizes the results of the exposure of LLC-PK1 cellsto plasma from control individuals, patients with Alzheimer's diseaseand multiple infarct dementia. There was a nearly fourfold increase inapoptotic index in LLC-PK1 cells that were exposed to Alzheimer'sdisease plasma (25.6±8.8%) as compared to control plasma (6.0±2.4%)P<0.001, and multiple infarct dementia plasma (6.5±2.3%), p<0.001. Therewas no significant difference in apoptotic index between control plasmaand multiple infarct dementia plasma, p>0.05. As noted in FIG. 3B,apoptotic index increased progressively as the time of incubation withAlzheimer's disease plasma increased, peaking at 2 h with an apoptoticindex of 16.1±0.3%. Diluting Alzheimer's disease plasma in a range of1:2 to 1:100 revealed a maximum apoptotic index of 12.4±0.2% at 1:5dilution of plasma with medium (FIG. 3A). There was no correlationbetween apoptotic index and the medications the patients had been takingat the time of study or the MMSE scores in Alzheimer's disease. TABLE 3Apoptotic index in LLC-PK1 cells exposed to control, Multi-Infarctdementia, and Alzheimer's disease patient's plasma AI (%) Alzheimer'sPlasma 25.6 ± 8.8  MID Plasma 6.5 ± 2.3 Control Plasma 6.0 ± 2.4

[0126]FIG. 4 shows internucleosomal DNA cleavage in LLC-PK1 cells thathad been exposed to plasma from patients with Alzheimer's disease (AD)for intervals of 2, 3, 4, and 5 days. Maximum DNA fragmentation was 4days after exposure to AD plasma and showed the characteristic 180 bpspacing. Control (NC) plasma did not exhibit a ladder even afterincubation for 5 days.

[0127] As noted in Table 4, elimination of calcium from the incubatingmedium, fetal bovine serum and plasma or incubation with 200 uMcycloheximide resulted in inhibition of apoptosis by Alzheimer's diseaseplasma, suggesting that apoptosis in the system is dependent on thelevel of extracellular calcium and protein synthesis. There was noinhibition of apoptosis at the lower concentrations of cycloheximide. Ina separate group of experiments, heating Alzheimer's disease and controlplasma at 56° C. for 30 min., which deactivated complement, did notalter the apoptotic activity, table 4. However, boiling the Alzheimer'sdisease plasma at 100° C. for 5 min. resulted in AT that was notdifferent from control plasma. Moreover, freezing and thawing the plasmafrom −70° C. to room temperature at least three times decreasedapoptotic activity, table 4. These data suggest that the apoptoticfactor in Alzheimer's disease plasma is a protein. TABLE 4Characteristics of Apoptotic Factor Alzheimer's Plasma Control PlasmaAI(%) AI(%) No treatment 25.6 ± 8.8  6.0 ± 2.4 56° C., 30 min. 19.5 ±2.7  8.3 ± 2.5 100° C., 5 min. 4.7 ± 2.2 8.7 ± 1.2 Cycloheximide (200μM) 7.7 ± 1.6 6.3 ± 1.5 (0.2-20 μM) 28.7 ± 4.0  5.0 ± 0.0 Ca⁺⁺ freemedium 6.3 ± 1.6 4.0 ± 0.7 Freeze and thaw 6.0 ± 2.4 5.3 ± 1.5

[0128] To exclude the possibility that the factor is β-amyloid, cellswere incubated with 0.1, 10 and 50 uM of β-amyloid dissolved in media.No detectable apoptotic activity was observed even at 50 uM,AI=6.1±3.1%. To eliminate the possibility that TNF-α might be theapoptotic factor, control and Alzheimer's disease plasma were quantifiedfor the presence of TNF-α by ELISA (Quantikine). The levels of TNF-α incontrol and Alzheimer's disease plasma were less than the lowest levelof detection by the ELISA kit of 0.3 pM. We achieved a standard curvewith the ELISA with TNF-α standards and blocked the reaction, utilizingTNF-α antibody. We also tested the effect of TNF-α on LLC-PK1 cells at5, 50, 500, and 3000 pM for 2 h and found that doses as high as 50 pMyielded background levels of apoptosis, AI=6%. On a Western blot boththe control and Alzheimer's disease plasma resulted in no signal, whilea positive control of 100 ng TNF-α yielded a positive signal. A similarsituation occurred with interleukin-1β. Both control and Alzheimer'sdisease plasma had undetectable levels of interleukin 1β by ELISA.

[0129]FIG. 5 depicts the protein profile from each individual step ofpurification on a Affi-Gel-Blue-Gel column. The highest AI of 21% wasfound in the 0.5 M NaCl eluate. No activity was found in the load andwash fraction and only a modest activity was noted in the 2M NaCleluate, AI of 6% vs. 9%, respectively. The 2M NaCl eluate was mainlycomposed of albumin. The active fraction (0.5 M NaCl eluate) wasdialyzed overnight in a 10 kDa m.w. cut-off membrane at 4° C. against 10mM phosphate buffer, pH 7.1 to remove salt. Isoelectric focusing wasperformed on this protein fraction at a pH gradient of 3-10, FIG. 6A.Fractions within clearly defined protein peaks were pooled and dialyzedto remove ampholyte, followed by a TUNEL assay to monitor for apoptoticactivity. Dialysis with a 10 kDa m.w. cut-off membrane demonstratedretention of apoptotic activity in the dialysis bag, suggesting that thesize of the protein exceeded 10 kDa. The highest AI of 29.4% was notedin fraction 2 of the pooled samples and isoelectric focusing repeatedonly on this active fraction at a narrow range pH gradient of 4-6, FIG.6B. The active fraction with an AI of 22% was noted in fraction 2 ofthis additional purification step. The pI range of both fractions was4.7-5.5.

EXAMPLE 2 Isolation and Identification of Prostaglandin D₂ Synthase

[0130] Eleven liters of urine were collected from a patient sufferingfrom renal salt wasting syndrome. The protein was precipitated from theurine with 80% ammonium sulfate and centrifuged to get a pellet. Aportion of the pellet was dissolved in 25 mM Tris.HCl at pH 7.5 and thendialyzed overnight in the same buffer in a 10 kDa cutoff membrane. Thedialyzed proteins were then loaded onto a High-Trap Q Sepharose column.The proteins were eluted off this column with 0.5M NaCl and 1.0 M NaClin several fractions. These fractions were then dialyzed in a phosphatebuffer at pH 7.1. Subsequently the fractions were assayed for theirability to induce apoptosis (see Experimental Details) and the activitywas found in the 0.5M NaCl. Following this, isoelectric focusing (seeExperimental Details) was performed from pH 3 to 10 and fractions werecollected. The active fraction (pH 4.8-5.5) was further purified byHPLC-C₁₈ column. The active fraction from this column was found in asingle peak. The active fraction was placed on SDS PAGE gel and proteinswith molecular weight of 29, 32, 33, and 42 Kd were eluted from the geland assayed for activity. The activity was found to be associated withthe 32 Kd band.

[0131] Following the above procedure, the 32 Kd band was sequenced andfound to contain 2 proteins, one of which is α₁ microglobulin. Since α₁microglobulin was found to have no apoptotic activity, it was absorbedon a protein A column to which the al microglobulin-specific antibodywas attached. The result was a pure 23-29 kD₂ band protein as seen on anSDS PAGE gel. The single 23-29 kD₂ protein was transferred from SDS-PAGEgel to a protein sequencing membrane and sequenced. With two separateanalyses based on the first 20 N terminal amino acids, the apoptoticfactor was positively identified as prostaglandin D₂ synthase whichsequence was described by Nagata et al. (Proc. Natl. Acad. Sci. USA,88:4020-4024; 1991).

EXAMPLE 3 Modulation of the Synthesis of -Δ¹²Prostaglandin J₂

[0132] Prostaglandin D₂ synthase is an enzyme involved in the-Δ¹²Prostaglandin J₂ synthesis pathway. The following is an illustrationof this pathway.

[0133] Prostaglandin D₂ Synthase increased apoptosis of human kidneyproximal tubule cells in culture about four times above control (seeExperimental Details). To find out which prostaglandin is responsiblefor inducing apoptosis, the different prostaglandins were tested fortheir ability to induce apoptosis in kidney proximal tubule cells.-Δ¹²Prostaglandin J₂ was found to be the only prostaglandin listed abovethat induces apoptosis. It induced apoptosis to the same degree asProstaglandin D₂ Synthase. Also, Prostaglandins E, H and D did notincrease apoptosis.

[0134] Adding Indomethacin, which blocks cyclo-oxygenase reduces theprostaglandins downstream and inhibited apoptosis to baseline.Furthermore, the simultaneous addition of Indomethacin and ProstaglandinD₂ Synthase did not increase apoptosis above baseline. In addition, thedeactivation of Prostaglandin D₂ Synthase by N-Ethyl Maleimide inhibitedapoptosis. The combination of Indomethacin, Prostaglandin D₂ Synthaseand Δ¹²Prostaglandin J₂ increased apoptosis and so did the addition ofΔ¹²Prostaglandin J₂ to Indomethacin increased apoptosis.

[0135] In combination, these results indicate that prostaglandin D₂synthase increases apoptosis by increasing the production ofprostaglandin D₂ which necessarily results in the production of-Δ¹²Prostaglandin J₂. It is therefore the activity of -Δ¹²prostaglandinJ₂ that clinical techniques should seek to reduce. Indirect methods suchas reducing activity of prostaglandin D₂ synthase or reducing any“upsteam” synthesis of -¹²prostaglandin J₂, or a precursor thereto, isquite useful. Increased catabolism of -Δ¹²prostaglandin J₂, its upstreamprecursors, or enzymes involved in its synthesis is also expected to beeffective. Naturally, direct inhibition of -Δ¹²prostaglandin J₂ activity(e.g. using a -Δ¹²prostaglandin inhibitor, e.g. a receptor antagonist)may also provide therapeutic effect.

EXAMPLE 4 Production of Antibodies to Prostaglandin D₂ Synthase

[0136] EST's homologous to mRNA for the glutathione independent PGD2Swere obtained from ATCC and assembled into a full-length cDNA and apremature stop codon mutation corrected. The full-length cDNA wasinserted into a bacterial expression vector, pMAL-C2, joining the vectorencoded carrier protein to the PGD2S coding sequence at the signalpeptidase cleavage site. Purified recombinant fusion protein waspurified by affinity chromatography, cleaved with factor Xa, and thecarrier protein separated from the recombinant factor by ion exchangechromatography. One milligram of purified recombinant factor was mixedwith Titermax adjuvant and injected intradermally in a New Zealand whiterabbit. Five weeks later, the rabbit was boosted with another milligramof recombinant factor in adjuvant and serum collected 10 days later.Polyclonal antisera from this rabbit was able to detect 1 nanogram ofreduced recombinant factor in a Western blot. This antisera also reactedin a Western blot with PGD2S from a natural source and which had thesame MW as described in the literature.

[0137] Although the present invention has been described in relation toparticular embodiments thereof, many other variations and modificationsand other uses will become apparent to those skilled in the art. Thepresent invention therefore is not limited by the specific disclosureherein, but only by the appended claims.

What is claimed is:
 1. A diagnostic kit for detecting the presence ofprostaglandin D₂ synthase in a sample, said kit comprising antibodies tosaid prostaglandin D₂ synthase, and means for measuring prostaglandin D₂synthase:anti-prostaglandin D₂ synthase immunocomplexes.
 2. The kit ofclaim 1, wherein said measuring means comprises an enzyme labelledanti-immunoglobulin and a chromogenic substrate for said enzyme label.3. The kit of claim 1, wherein said antibodies are immobilized on asubstrate.
 4. The kit of claim 1, wherein said kit is for use indetection of Alzheimer's disease, and further includes a comparisonstandard for correlating said measurement to likelihood a patient fromwhom said sample was taken is afflicted with Alzheimer's disease.
 5. Thekit of claim 1, wherein said kit is for detection of renal salt wastingsyndrome, and further includes a comparison standard for correlatingsaid measurement to likelihood a patient from whom said sample was takenis afflicted with renal salt wasting syndrome.